Elevated homocysteine (Hcy) in the blood is an established risk factor for cardiovascular disease. Increases in dietary folate and B6 have been shown to lower Hcy levels. There are however, certain groups with other health problems where Hcy remains elevated, these include heart transplant recipients, diabetics, women with preclampsia or retarded fetal growth, end stage renal disease and Parkinson's disease. The aim of this proposal is to elucidate how nutritional insufficiency of folate and B6 affect the pathways of Hcy metabolism in mammalian cells. There are four specific aims: (1) the development of rapid enzyme-based assays for 5,10-methyleneTHF, B6 vitamers and homocysteine; (2) to determine the direction of flux of 1-carbon (1-C) groups in the cytosol and mitochondria of cells in culture, with special emphasis on serine hydroxmethyltransferase (SHMT); (3) to determine the role of mitochondria in the supply of 1-C groups to the cytosol; and (4) to determine the relationship of folate pools and metabolic levels of homocysteine with several different cell lines when either folate or B6 are limiting growth factors. Three hypotheses will be tested, which are: (1) that the role of cytosolic SHMT is not to generate 1-C units but to regulate the levels of glycine and 5,10-methyleneTHF in the cytosol; (2) that 1-C groups used by the cytosol are generated by the mitochondria as formate; and (3) Hcy levels are related to the level of 5,10-methyleneTHF in the cytosol.